This invention relates to substituted 3-quinolinecarbonitrile compounds as well as the pharmaceutically acceptable salts thereof. The compounds of the present invention inhibit the action of certain enzymes known as protein kinases. Protein kinases play a vital role as key regulators of a variety of critical cell functions. These enzymes function by catalyzing the transfer of a phosphate group from ATP to amino acid residues of substrate proteins. Tumorigenesis has been linked to the aberrant function of protein kinases and protein kinases are of particular interest as potential targets for anticancer agents.
Protein kinases are a class of enzymes that catalyze the transfer of a phosphate group from ATP to a tyrosine, serine, threonine, or histidine residue located on a protein substrate. Protein kinases clearly play a role in normal cell growth. Many of the growth factor receptor proteins function as kinases and it is by this process that they effect signaling. The interaction of growth factors with these receptors is a necessary event in normal regulation of cell growth. However, under certain conditions, as a result of either mutation or over expression, these receptors can become deregulated. Specific protein kinases have been implicated in diverse conditions including cancer (Traxler, P. M., Exp. Opin. Ther. Patents, 8, 1599 (1998); Bridges, A. J., Emerging Drugs, 3, 279 (1998)), restenosis (Mattsson, E., Trends Cardiovas. Med. 5, 200 (1995); Shaw, Trends Pharmacol. Sci. 16, 401 (1995)), atherosclerosis (Raines, E. W., Bioessays, 18, 271 (1996)), angiogenesis (Shawver, L. K., Drug Discovery Today, 2, 50 (1997); Folkman, J., Nature Medicine, 1, 27 (1995)) and osteoporosis (Boyce, J. Clin. Invest., 90, 1622 (1992)).
One signaling pathway found in all eukaryotic organisms is the Ras-MAPK module, which is comprised of the Ras/Raf/MEK (mitogen-activated protein kinase)/MAPK (mitogen-activated protein kinase) signaling cascade. This key pathway is involved in transmitting signals from growth factors and hormones at the extracellular compartment into the cytosol and to transcription factors in the nucleus. Alteration of the Ras-MAPK pathway is associated with the formation of certain human tumors.
Components of the Ras-MAPK Signaling Cascade
The Ras/Raf/MEK/MAPK signaling cascade is activated by GTP loading of Ras, which occurs in response to stimuli from cell surface receptors (Malumbres, M., Barbacid, M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003, 3: 459-65; Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 2003, 3: 11-22; Bos, J. L. Ras oncogenes in human cancer. A review. Cancer Res 1989, 49: 4682-4689). Ras is a member of a large family of small (21 kDa) GTPases that act as molecular switches in the regulation of cell growth, differentiation, survival, and apoptosis (Malumbres, M., Barbacid, M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003, 3: 459-65; Downward, J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 2003, 3: 11-22; Bos, J. L. Ras oncogenes in human cancer: A review. Cancer Res 1989, 49: 4682-4689; Shields, J. M., Pruitt, K., McFall, A. et al. Understanding Ras: ‘it ain't over til it's over’. Trends Cell Biol 2000, 10: 147-154). Ras is initially defined as a component of oncogenic murine retroviruses (Ellis, R. W., Defeo, D., Shih, T. Y. et al. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature 1981, 292: 506-511), and it became the focus of intensive research in the early 1980s when the connection between mutant forms of Ras and human cancer was established (Ellis, R. W., Defeo, D., Shih, T. Y. et al. The p21 src genes of Harvey and Kirsten sarcoma viruses originate from divergent members of a family of normal vertebrate genes. Nature 1981, 292: 506-511; Perucho, M., Goldfarb, M., Shimizu, K., Lama, C., Fogh, J. and Wigler, M. Human-tumor-derived cell lines contain common and different transforming genes. Cell 1981, 27: 467-476; Santos, E., Martin-Zanca, D., Reddy, E. P., Pierotti, M. A., Della Porta, G., Barbacid, M. Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient. Science 1984, 223: 661-664). Ras mutations that lead to oncogenic activation occur primarily in two hotspots: Gly12 and Gln61 (Krengel, U., Schlichting, L., Scherer, A. et al. Three-dimensional structures of H-ras p21 mutants: Molecular basis for their inability to function as signal switch molecules. Cell 1990, 62: 539-548). The mutational changes at these residues found in human cancers impede the GTP hydrolysis activity of Ras, thus causing Ras to remain in the GTP-bound or “on” conformation. With this finding, a better appreciation of the molecular basis of aberrant signaling associated with ˜30% of all human cancers are obtained.
There are three Ras isoforms associated with human cancer, H-Ras, N-Ras, and K-Ras; and of these, 95% are due to K-Ras mutations. Ras proteins are closely related, having 85% amino acid identity with a 20 amino acid variable region at the carboxy terminus (Lowy, D. R. and Willumsen, B. M. Function and regulation of ras. Annual Review of Biochemistry 1993, 62: 851-891). A Cysteine residue occurs in all Ras proteins after the variable region, and this is the site of post-translational modification of Ras by addition of a farnesyl isoprenoid lipid (Hancock, J. F., Magee, A. I., Childs, J. E. and Marshall, C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell 1989, 57: 1167-1177). All Ras proteins are further modified by proteolysis of the three carboxy terminal amino acids and subsequent methylation of the new carboxy terminus. These modifications stabilize Ras interaction with the inner cell membrane where it must reside to form a multi-protein complex with Raf, MEK, MAPK and scaffold proteins to activate signaling in the Ras-MAPK module (Kolch, W. Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochemical J 2000, 351 Pt 2: 289-305; Kolch, W. Ras/Raf signalling and emerging pharmacotherapeutic targets. Expert Opin Pharmaco 2002, 3: 709-718).
As the consequences of mutated Ras function were elucidated, this attracted sufficient attention from numerous pharmaceutical laboratories to initiate discovery and development programs for small molecule inhibitors of this signaling protein. The goal of these programs is to bring forward inhibitors of aberrant Ras signaling that have minimal associated toxicities. Numerous Ras inhibitors have been described, and they impair Ras-MARK signaling by blocking proper post-translational modification of Ras by protein farnesyl transferase, thereby preventing membrane localization. These Ras inhibitors have shown good pre-clinical efficacy, and are currently being evaluated in clinical trials. The sequential downstream kinase effectors of Ras, namely Raf, MEK, and MAPK are in addition viewed as equally attractive targets for the pharmacological intervention of cancer.
Rat proteins are fairly homologous (˜60%). These 66-84 kDa serine/threonine kinases include A, B, and C isoforms (C-Raf=Raf1, c-Raf), which coexist in many cell types and activate MEK (Kolch, W. Ras/Raf signalling and emerging pharmacotherapeutic targets. Expert Opin Pharmaco 2002, 3: 709-718; Chong, H., Vikis, H. G., Guan, K.-L. Mechanisms of regulating the Raf kinase family. Cell Signal 2003, 15: 463-469; Chong, H., Lee, J., Guan, K. L. Positive and negative regulation of raf kinase activity and function by phosphorylation. EMBO J 2001, 20: 3716-3727). Three conserved regions occur in Raf proteins: CR1 which is at the N-terminal and contains a Ras binding domain (RBD) and a cysteine rich domain (CRD); CR2 which contains a serine/threonine rich region; and CR3 which contains the catalytic kinase domain. GTP-loaded Ras recruits Raf to the inner cell membrane. This is crucial for Raf activation, though activation is a complex process not yet fully understood. For example, there are at least 13 regulatory phosphorylation sites on C-Raf (Fabian, J. R., Daar, I. O., Morrison, D. K. Critical tyrosine residues regulate the enzymatic and biological activity of raf-1 kinase. Mol Cell Biol 1993, 13: 7170-7179; Marais, R., Wynne, J., Treisman, R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 1993, 73: 381-393; Morrison, D. K., Heidecker, G., Rapp, U. R. and Copeland, T. D. Identification of the major phosphorylation sites of the Raf-1 kinase. J Biol Chem 1993, 268: 17309-17316; Mason, C. S., Springer, C. J., Cooper, R. G., Superti-Furga, G., Marshall, C. J., Marais, R. Serine and tyrosine phosphorylations cooperate in Raf-1 but not B-Raf activation. EMBO J 1999, 18: 2137-2148; Dhillon, A. S., Meikle, S., Yazici, Z., Eulitz, M., Kolch, W. Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J 2002, 21: 64-71; Kolch, W. To be or not to be: a question of B-Raf? Trends Neurosci 2001, 24: 498-600; Abraham, D., Podar, K., Pacher, M. et al. Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem 2000, 275: 22300-22304; Yeung, K., Seitz, T., Li, S. et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 1999, 401: 173-177; Chang, F., Steelman, L. S., Lee, J. T. et al. Signal transduction mediated by the RasRafMEKERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention. Leukemia 2003 17:1263-1293). Not only GTP-loaded Ras, but also various kinases (e.g. C-Tak1, PAK, PKC, PKA, Src), phosphatases (PP1, PP2A), adapter proteins, and scaffold proteins (KSR) are implicated in full Raf activation (Dhillon, A. S. and Kolch, W. Untying the regulation of the Raf-1 kinase. Arch Biochem Biophys 2002, 404: 3-9; Morrison, D. K. KSR: a MAPK scaffold of the Ras pathway? J Cell Sci 2001, 114: 1609-1612; Baccarini, M. An old kinase on a new path: Raf and apoptosis. Cell Death Differ 2002, 9: 783-785). The three Raf isoforms differ in their ability to interact with Ras isoforms, to activate MEK, and to transform rodent fibroblasts in vitro (Pritchard, C. A., Bolin, L., Slattery, R., Murray, R., McMahon, M. Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase gene. Curr Biol 1996, 6: 614-617). The B-Raf isoform in all cases is the most active followed by C-Raf, and then A-Raf.
A, B, and C Raf knockout mice have been described (Wojnowski, L., Zimmer, A. M., Beck, T. W. et al. Endothelial apoptosis in Braf-deficient mice. Nat Genet 1997, 16: 293-297; Huser, M., Luckett, J., Chiloeches, A. et al. MEK kinase activity is not necessary for Raf-1 function. EMBO J 2001, 20: 1940-1951; Murakami, M. S., Morrison, D. K. Raf-1 without MEK? Science's Stke: Signal Transduction Knowledge Environment 2001, 2001: PE3). B-Raf deficient embryos die at mid-gestation due to apoptotic cell death in endothelial cells leading to vascular hemorrhage. C-Raf deficiency causes mid-gestational death due to more diffuse apoptotic tissue effects. A-Raf deficient mice are born alive, but show neurological and intestinal defects. These divergent phenotypes show that Raf isoforms serve distinct functions in different tissues. These studies have shown that individual B-Raf and C-Raf survival functions cannot be performed by other Raf isoforms. They also demonstrated that normal levels of MAPK activation occurs in C-Raf deficient mouse cells, indicating that the anti-apoptotic function of C-Raf is not mediated by the MAPK cascade.
The anti-apoptotic function of C-Raf may be mediated by antagonism of apoptosis-stimulated kinase 1 (ASK-1). There is evidence to suggest that C-Raf impedes ASK-1 function via a protein-protein interaction that is not associated with C-Raf kinase activity (Chen, J., Fujii, K., Zhang, L., Roberts, T., Fu, H. Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc Natl Acad Sci USA 2001, 98: 7783-7788). Raf also can impede apoptosis in a kinase dependent manner. For example, Raf/MEK/MAPK signaling activates Rsk1, which in turn phosphorylates and inactivates BAD, a pro-apoptotic protein (Shimamura, A., Ballif, B. A., Richards, S. A., Blenis, J. Rsk1 mediates a MEK-MAP kinase cell survival signal. Curr Biol 2000, 10: 127-135). Additionally, C-Raf can be localized to the mitochondria by a Bcl-2 mediated process, where it can inactivate pro-apoptotic proteins by phosphorylation (Wang, H. G., Miyashita, T., Takayama, S. et al. Apoptosis regulation by interaction of Bcl-2 protein and Raf-1 kinase. Oncogene 1994, 9: 2751-2756). Raf anti-apoptotic effects are complex and will require further study to clarify. However, this characteristic of Raf enhances its appeal as a pharmaceutical target since a hallmark of cancer cells is resistance to apoptosis which at least in part is likely attributable to improper Raf activation (Herrera, R., Sebolt-Leopold, J. S. Unraveling the complexities of the Raf/MAP kinase pathway for pharmacological intervention. Trends Mol Med 2002, 8: S27-31).
As with Ras, oncogenic forms of Raf have been found to be components of transforming murine retroviruses (Mark, G. E., Rapp, U. R. Primary structure of v-raf: relatedness to the src family of oncogenes. Science 1984, 224: 285-289). Oncogenic Raf in murine retroviruses results from N-terminal deletions that remove the regulatory sequences that control Raf kinase activity. Most recently, a systematic human genome-wide screening effort to detect alterations in genes that control cell proliferation, differentiation, and death found activating B-Raf mutations in 66% of malignant melanomas (Davies, H., Bignell, G. R., Cox, C. et al. Mutations of the BRAF gene in human cancer. Nature 2002, 417: 949-954). Additionally, B-Raf mutations are observed at lower frequencies in a wide range of other cancers including colorectal, lung, breast and ovarian.
The significance of B-Raf mutations in colorectal tumors is extended in a subsequent study showing that mutations in either B-Raf or K-Ras (not both) are detected in a sample of colorectal tumors examined at all stages of development, including pre-malignant lesions (Rajagopalan, H., Bardelli, A., Lengauer, C., Kinzler, K. W., Vogelstein, B., Velculescu, V. E. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002, 418: 934). The frequency of B-Raf mutations in these colorectal tumors is 10%, whereas the K-Ras mutation frequency is 51%. Cumulative K-Ras/B-Raf mutation frequency in colorectal cancer is therefore 61%. In the case of melanoma, an examination of N-Ras mutation (the Ras isoform mutated in melanoma), together with B-Raf has shown a cumulative mutation frequency of 81% (Smalley, K. S. A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 2003, 104: 527-532). These statistics, when combined with the 2002 incidence of colorectal cancer (148,000) and melanoma (54,000) in the U.S. (American Cancer Society), make Raf a compelling pharmaceutical target.
MEK1 and MEK2 are expressed 43-46 kDa kinases activated by Raf phosphorylation of two serine residues (Ser217-Ser221). MEK1 and MEK2 are members of a larger family of dual specificity kinases (MEK1-7) that phosphorylate Threonine and Tyrosine residues within the TXY motif of various MAP kinases (Dhanasekaran, N., Premkumar Reddy, E. Signaling by dual specificity kinases. Oncogene 1998, 17: 1447-1455). MEK1 and MEK2 are encoded by distinct genes, but they have high homology (80%) within the C-terminal catalytic kinase domain and most of the N-terminal regulatory domain (English, J., Pearson, G., Wilsbacher, J. et al. New insights into the control of MAP kinase pathways. Exp Cell Res 1999, 253: 255-270). At the N-terminus of MEK1 and MEK2 there are 30 amino acids of divergent sequence that may direct differential interactions with both activators and substrates. The only known substrates for MEK1/MEK2 are the MAPK1 and MAPK2, which they phosphorylate on Thr202/183 and Tyr204/185, respectively.
MEK1 deficient mice have been described, and inactivation of MEK1 leads to embryonic lethality due to decreased placental vascularization during embryogenesis (Giroux, S., Tremblay, M., Bernard, D. et al., Embryonic death of Mek1-deficient mice reveals a role for this kinase in angiogenesis in the labyrinthine region of the placenta. Curr Biol 1999, 9: 369-372). MEK1 deficiency is not compensated for by MEK2. In contrast, MEK2 deficient mice are viable and fertile, with no morphological alterations (Bélanger, L. F., Roy, S., Tremblay, M. et al. Mek2 is dispensable for mouse growth and development. Mol Cell Biol 2003, 23: 4778-4787). These data demonstrate that MEK2 is not necessary for the normal development of mouse embryos, indicating that the loss of MEK2 can be compensated for (Bélanger, L. F., Roy, S., Tremblay, M. et al. Mek2 is dispensable for mouse growth and development. Mol Cell Biol 2003, 23: 4778-4787) by at least in part MEK1.
Oncogenic forms of MEK1 or MEK2 have not been described in retroviruses or human cancers. However, a MEK1 where Ser218 and Ser222 are both mutated to Asp is capable of causing oncogenic transformation of various rodent fibroblast cell lines (Mansour, S. J., Matten, W. T., Hermann, A. S. et al. Transformation of mammalian cells by constitutively active MAP kinase. Science 1994, 265: 966-970).
The MAPK components of the Ras-MAPK module have also been designated ERK1 and ERK2 (extracellular signal-regulated kinases). These MAPK isoforms (also designated p44 MAPK and p42 MAPK) are highly homologous (>80%), expressed 44-42 kDa serine/threonine kinases that are members of a larger gene family that includes ERK 1, 2, 3, 5, 7; JNK 1-3; and p38  and . Experimental data indicate that ERK1 and ERK2 are functionally equivalent (English, J. M., Cobb, M. H. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 2002, 23: 40-45). ERKs are activated by MEK phosphorylation of their TEY sequence; dual phosphorylation is required for activation, and in the case of ERK2 results in a >1000 fold increase in activity. Downstream substrates of ERK1/2 include cytoskeletal proteins, kinases, phosphatases, and transcription factors. The pleiotropic effects of MAPK activation on cell growth and differentiation are undoubtedly mediated through this diverse array of effectors.
No constitutively active MAP kinases are known, despite attempts at their genetic selection and site-directed mutagenesis. This failure suggests that cells cannot tolerate the continuous activation of MAP kinases. Among the kinase components of the Ras-MAPK signaling only the ERK2 atomic structure has been solved (Zhang, F., Strand, A., Robbins, D., Cobb, M. H., Goldsmith, E. J. Atomic structure of the MAP kinase ERK2 at 2.3 {acute over (Å)} resolution. Nature 1994, 367: 704-711).
The ERK1/ERK2 components of the Ras-MAPK module are the most abundant (˜106 molecules per cell). MEK also is relatively abundant in most cell types (˜3.5×105 molecules per cell), whereas Raf and Ras molecules are less abundant (˜2×104 per cell) (Ferrell, J. E., Jr. Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs. Trends Biochem Sci 1996, 21: 460-466). All MAPK molecules can become fully activated in cells where only 10-50% of Ras molecules are GTP bound. The predicted sensitivity of the Raf/MEK1/MAPK signaling cascade to inhibitors is: Raf>MEK1>MAPK (Huang, C. Y., Ferrell, J. E., Jr. Ultrasensitivity in the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 1996, 93: 10078-10083). This sensitivity profile results from the distributive (non-processive) mechanism of both Raf and MEK1 in which the rate of MEK1 activation depends on the concentration of Raf squared; and similarly the rate of MAPK activation is dependent on the concentration of MEK1 squared. To date, potent inhibitors of Raf and MEK, but not ERK, have been reported.